Field of the Invention
The invention relates to a droplet generator incorporated into a microfluidic chip. Specifically, the droplet generator generates droplets of an aqueous solution on a microfluidic chip with an air continuous phase.
Discussion of the Background
The detection of nucleic acids and the ability to perform biochemical assays and the like is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. Other biochemical assays, including be detection of proteins or other markers in a sample are relevant both to disease and disorder detection as well as environmental safety.
One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase Chain Reaction (“PCR”) is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies of the amplified DNA to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et at, eds., Academic Press Inc. San Diego, Calif. (1990),
Microfluidic chips are being developed for “lab-on-chip” devices to perform biochemical assays including in vitro diagnostic testing. The largest growth area is in molecular biology where DNA amplification is performed in the sealed channels of the chip. Optical detection devices are commonly used to measure the increasing amplicon product over time (Real Time PCR) and/or to perform a thermal melt to identify the presence of a specific genotype (High Resolution Thermal Melt).
Droplet PCR is well known in the art, and has previously taken the form of an aqueous droplet surrounded by an immiscible fluid, such as an oil, a fluorinated liquid, or any other non-aqueous or hydrophobic solvent. However, droplet PCR using an oil phase has some drawbacks. Use of a water-in-oil droplet requires additional materials in comparison to standard PCR (i.e., oils, surfactants, etc.), and proteins can be denatured at the oil-water interface due to their contact with the oil, which can lead to irreversible protein adsorption onto the surface of a microfluidic channel. Further, the viscosity of oil requires slower flowrates than can be achieved with other materials.
Droplet PCR has particularly been used in lab-on-chip applications, both in flow-through microfluidic channels (biochemical reactions may be performed on the samples either while stationary or while flowing through the channel) and in microfluidic systems incorporating traps in which the droplets can be contained in the microfluidic system. For instance, hydrodynamic traps are described in Bithi and Vanapaili (“Behavior of a train of droplets in a fluidic network with hydrodynamic traps”, Biomicrofluidies 4, 044110 (2010)).
Bithi and Vanapalli describe the use of both passive and active methods for trapping and storing droplets in microfluidic systems. In some instances, passive trapping is preferred as it is more scalable to allow multiplexing than active trapping may be. Bithi and Vanapalli describe two methods of passive trapping, direct and indirect trapping, which are based on the hydrodynamic resistance of an upper and lower branch of a microfluidic system containing a repetitive series of loops, as is shown in FIG. 1. As noted in FIG. 1, this system of trapping droplets is designed to work with a water-in-oil system, as described above. The effectiveness of trapping droplets in such a system is dependent on droplet size and droplet spacing, requiring precise control of the water-in-oil droplet formation Oil flow rate is a key factor in the performance of such a system, and system paramaters would need to be optimized for the specific oil or other surfactant used in creating the droplets.
Accordingly, a need exists in the art for alternate systems and methods of preparing droplets for use on microfluidic chips that overcome these drawbacks.